Identification of 12/15-lipoxygenase as a suppressor of myeloproliferative disease

Abstract

Though Abl inhibitors are often successful therapies for the initial stages of chronic myelogenous leukemia (CML), refractory cases highlight the need for novel molecular insights. We demonstrate that mice deficient in the enzyme 12/15-lipoxygenase (12/15-LO) develop a myeloproliferative disorder (MPD) that progresses to transplantable leukemia. Although not associated with dysregulation of Abl, cells isolated from chronic stage 12/15-LO-deficient (Alox15) mice exhibit increased activation of the phosphatidylinositol 3-kinase (PI3-K) pathway, as indicated by enhanced phosphorylation of Akt. Furthermore, the transcription factor interferon consensus sequence binding protein (ICSBP) is hyperphosphorylated and displays decreased nuclear accumulation, translating into increased levels of expression of the oncoprotein Bcl-2. The ICSBP defect, exaggerated levels of Bcl-2, and prolonged leukemic cell survival associated with chronic stage Alox15 MPD are all reversible upon treatment with a PI3-K inhibitor. Remarkably, the evolution of Alox15 MPD to leukemia is associated with additional regulation of ICSBP on an RNA level, highlighting the potential usefulness of the Alox15 model for understanding the transition of CML to crisis. Finally, 12/15-LO expression suppresses the growth of a human CML-derived cell line. These data identify 12/15-LO as an important suppressor of MPD via its role as a critical upstream effector in the regulation of PI3-K-dependent ICSBP phosphorylation.

Figure 1.

Decreased survival and marked splenomegaly in 12/15-LO–deficient mice. (a) Kaplan-Meier plot of 102 wild-type (B6) and 113 Alox15 mice representing a nearly 1:1 ratio of males/females. (b) Representative spleens of 10–12-wk-old wild-type, asymptomatic (Asx), and moribund Alox15 mice. (c) Mean wet weights ± SD of the spleens shown in b (n = 18 for B6 and Asx Alox15 mice; n = 9 for moribund Alox15 mice). *, P < 0.0001.

Figure 2.

Disrupted architecture and increased myeloid cells in the spleens of 12/15-LO–deficient mice. (a) H &E staining of 8-μm frozen sections of spleens from wild-type (n ≥ 10), Asx Alox15 (n ≥ 10), and moribund Alox15 mice (n = 7). WP, white pulp; RP, red pulp. Bar, 50 μm. Fluorescence-activated flow cytometric analysis of (b) B cell populations, (c) T cell populations, and (d) myeloid populations in isolated splenocytes of representative samples gated on live cells. Percentages for each quadrant are indicated. Mac-1/GR-1 double-positive cells increased from a range of 1.8–8.2% in wild-type (n = 11) to 15–24% and 35–70% in the spleens of Alox15 mice in the chronic (n = 9) and crisis (n = 4) phases, respectively. (e) Differential counts of cytospin preparations of splenocytes from asymptomatic (Asx) and moribund Alox15 mice versus wild-type controls.

Figure 3.

Increased myeloid cells in the bone marrow, blood, and lymph node of 12/15-LO–deficient mice. (a, top) Representative (n = 6) wild-type (B6) and Alox15 bone marrow cells. Note the large megakaryocyte (black arrowhead) in the asymptomatic (Asx) Alox15 cells. Green arrowheads indicate myeloblasts. Note the presence of basophilic cytoplasm, prominent nucleoli, undefined granules, and the absence of Auer rods (Auer rods would support a diagnosis of acute myeloid leukemia; reference 56). Bar, 50 μm. (a, middle) Representative flow cytometric analysis of Mac-1 and Gr-1 expression in bone marrow. Percentages for each quadrant are indicated. Double-positive ranges: C57BL/6, 35.9–52.7% (n = 7); Asx Alox15, 43.8–61.2% (n = 7); and moribund Alox15, 61.9–90.1% (n = 5). (a, bottom) Gr-1 and CD34 expression in bone marrow. Percentages for each quadrant are indicated. Double-positive ranges: C57BL/6, 4.5–9.1% (n = 5); Asx Alox15, 11.2–17.9 (n = 4); and moribund Alox15, 25.8–33.1% (n = 3). (b, top) C57BL/6 and Alox15 blood smears. Note the platelet anisocytosis and micromegakaryocytosis often found in CML. Black arrows point to examples of basophils, and green arrowheads indicate example blast cells. Bar, 50 μm. (b, bottom) Flow cytometric analysis of Gr-1 expression in blood leukocytes from 25-wk-old B6, Asx Alox15, and moribund Alox15 mice. Percentages of Gr-1⁺ cells are indicated. Gr-1⁺ ranges: B6, 42.5–51% (n = 5); Asx Alox15, 62–79.2% (n = 5); and moribund Alox15, 68.2–90.% (n = 3). (c, top) H &E-stained sections of wild-type and chronic Alox15 lymph node. Arrowheads point to pseudo-Gaucher–like cells. (c, bottom) Immunohistochemical staining for Gr-1⁺ cells in B6 and Alox15 lymph node. Bar, 50 μm.

Figure 4.

12/15-LO suppresses proliferation and survival in mouse and human cells. (a) Flow cytometric analysis of propidium iodide–stained wild-type (B6) and chronic Alox15 (Chr) sorted Gr-1⁺ splenocytes. (b) Quantification of data shown in panel a. *, P = 0.01 compared with B6 (n = 3). (c) The percentage of apoptotic cells in B6 and Alox15 myeloid cells from 10–20-wk-old B6 and Alox15 mice after 24-h culture was quantified using propidium iodide staining. *, P = 0.002 compared with B6 (n = 4). (d) Immunoblot of 12/15-LO in K562 cells transfected with a vector control or mouse 12/15-LO, as compared with sorted B6 splenic macrophages (Mac). An anti–human 12/15-LO antibody that cross reacts with mouse (not depicted) was used. Samples were normalized for protein concentration. (e) The number of viable K562 cells 24 h after transfection (Tx) with 12/15-LO or vector control (Ctl) in the presence of 10 μM PD146176 (PD146) or DMSO vehicle control was quantified using trypan blue exclusion. *, P = 0.0268 compared with Ctl DMSO (n = 3); #, P = 0.0256 compared with 12/15-LO DMSO (n = 3). (f) Proliferation index after 24 h of transfected K562 cells, as measured by the fold increase in viable cells treated with 10 μM PD146176 or DMSO vehicle control. *, P = 0.0149 compared with Ctl DMSO (n = 3); #, P = 0.005 compared with 12/15-LO DMSO vehicle. All error bars represent means ± SD.

Figure 5.

The MPD in 12/15-LO–deficient mice is cell autonomous. (a) Increased splenic weight, (b) representative H &E-stained cryostat sections of spleens, and (c) cytospins of splenocytes from 8–10-wk-old wild-type mice injected via the tail vein with 0.5 × 10⁶ (Alox15 crisis) or 10⁷ (Alox15 chronic and C57BL/6) splenocytes or 2 × 10⁶ bone marrow cells, monitored every other day, and killed at 6 or 10 wk after transfer. Bar, 50 μm. (d) 2% agarose electrophoresis of PCR products for the neomycin cassette performed on DNA extracted from spleens taken from mice transplanted with B6 (B6 Trx) or Alox15 (ATrx) cells versus Alox15 tail DNA positive control. (e) Table summarizing transfer experiments performed as described in panel a. Splenic distortion was assessed blindly. All error bars represent means ± SD.

Figure 6.

12/15-LO–deficient MPD may be due in part to the loss of lipid mediators. Mass spectrometric or (in the case of lipoxin A₄) ELISA analysis of the release per mg of tissue of (a) 5(S)-HETE, (b) lipoxin A₄, (c) 12(S)-HETE, (d) 13(S)-HODE, and (e) 15(S)-HETE in PMA and ionomycin-stimulated unperfused spleens (n = 4). *, P = 0.0058; #, P = 0.0006. Shaded and open bars represent C57BL/6 and chronic Alox15 mice, respectively. (f) 24-h viability in Alox15 chronic(Chr) or K562 cells treated overnight with 60 μM 12(S)-HpETE, a direct 12/15-LO product. *, P = 0.013 (n = 3); #, P = 0.003 (n = 3). All error bars represent means ± SD.

Figure 7.

Alox15 MPD is Abl independent. Quantification of (a) cycling and (b) apoptotic K562 cells treated for the indicated times with 16 μM STI571 or DMSO vehicle control. *, P = 0.036 compared with DMSO control (n = 3); #, P = 0.042 compared with DMSO control (n = 3). Quantification of (c) cycling and (d) apoptotic cells in C57BL/6 wild-type or chronic Alox15 (Chr) splenocytes treated for the indicated times with 16 μM STI571 or DMSO vehicle control (n = 3). (e) Crk immunoprecipitation (IP) and immunoblot for phosphotyrosine (P-tyr) or Crk loading control of cytoplasmic lysates prepared from B6 and chronic Alox15 splenocytes (n = 4). (f) Quantification of the data shown in e. All error bars represent means ± SD.

Figure 8.

ICSBP defect and enhanced Bcl-2 expression in cells from Alox15 mice. (a) Immunoblot of nuclear extracts prepared from wild-type (B6), chronic Alox15 (Chr), and crisis Alox15 (Cri) splenocytes using antibodies specific for ICSBP and normalized to total protein, as confirmed by coomassie stain (loading). (b) Real-time PCR analysis of ICSBP mRNA expression levels in wild-type, chronic Alox15, and crisis Alox15 splenocytes. *, P = 0.0115 and 0.0284 compared with B6 and Chr, respectively. (c) Real-time PCR of sorted myeloid splenocytes isolated from C57BL/6 and chronic stage Alox15 mice. (d) Representative immunoblot of nuclear (above line) and cytoplasmic (below line) extracts prepared from sorted wild-type and Alox15 splenocytes for ICSBP, 12/15-LO, Bcl-2, and the loading controls histone 2b and total actin. The results are representative of three experiments. All error bars represent means ± SD.

Figure 9.

PI3-K regulates Alox15 myeloproliferative cell survival and ICSBP phosphorylation and nuclear accumulation. (a) Immunoblot of ICSBP immunoprecipitated from wild-type (B6) and chronic Alox15 (Chr) splenocyte total lysates. (b) Constitutive levels of phosphorylated Akt in bone marrow myeloid cells cultured for 5 d in GM-CSF. (c) Splenocytes were cultured for 6 h in 16 μM Ly296002 or DMSO vehicle control, and samples were prepared and analyzed as in panel a. (d) Quantification of the data shown in c (n = 3). *, P = 0.0012 compared with B6 DMSO; #, P = 0.0081 compared with Chr DMSO. (e) Nuclear extracts of cells treated as in c. (f) Quantification of the data shown in e (n = 3). *, P = 0.0052 compared with B6 DMSO; #, P = 0.0155 compared with Chr DMSO. (g) Real-time PCR of Bcl-2 transcripts in sorted myeloid splenocytes from C57BL/6 and Alox15 cells. *, P = 0.001; #, P = 0.02. (h) Quantification of splenocyte viability by trypan blue exclusion after 24-h culture of B6 and Chr splenocytes with Ly294002 or DMSO control (n = 3). *, P = 0.0326 compared with B6 DMSO; #, P = 0.006 compared with B6 DMSO. All error bars represent means ± SD.

Figure 10.

Potential mechanisms by which 12/15-LO may regulate ICSBP. Absence of 12/15-LO leads to increased PI3-K pathway activation, possibly via loss of inhibition of activators such as PDK1 or gain of activity of PTEN. PI3-K–dependent phosphorylation of ICSBP may reduce nuclear levels by diminished nuclear translocation, increased nuclear turnover, or both.